Leucine rich repeat kinase 1 (Lrrk1) belongs to ROCO family proteins and contains three ankyrin repeat, seven leucine-rich repeat, a GTPase-like domain of Roc (Ras of complex proteins), COR (C-terminal of Roc) domain, and a serine/threonine kinase domain that is regulated by GTP binding to the Roc domain. Lrrk2 contains most domains of ROCO family protein and an extra Lrrk2 specific repeat, but lacks ankryrin repeat. Both Lrrk1 and Lrrk2 are ubiquitously expressed in multiple tissues. We recently found that mice with disruption of Lrrk1 gene displayed a severe phenotype of osteopetrosis caused by dysfunction of multinucleated cells while mice lacking Lrrk2 gene failed to show obvious skeletal phenotypes, indicating that Lrrk1 has a unique structure and function in bone cells that Lrrk2 cannot compensate for loss of Lrrk1. Little is known on the signaling pathways of Lrrk1 in osteoclasts. Our focus in this grant is to determine the structure and function of Lrrk1 i osteoclasts and identify potential Lrrk1 kinase targets and inhibitors. To this end, we propose two hypotheses in this study: 1) Lrrk1 regulates osteoclast functions via its kinase activation; 2) Lrrk1 phosphorylates serine/threonine residues of its specific substrate(s). To test the hypothesis 1, experiments are designed to examine the structure and function of full length Lrrk1 and truncated Lrrk1 on bone resorptive activity of mature osteoclasts on bone slices by in vitro pit assay. Homology models of human Lrrk1 kinase domain are used to dock small molecule inhibitors and the inhibition of drug-like compounds will be tested in vitro kinase and pi formation assays. To test the hypothesis 2, experiments are designed to enrich phosphor-peptides from wild type and Lrrk1 deficient osteoclasts via immunoaffinity purification by using phosphor-serine/phosphor-threonine motif antibodies, and identify Lrrk1 initial targets by liquid chromatography and tandem mass spectrometry (LC- MS/MS). The results of this application will advance our understanding of the molecular mechanisms of Lrrk1 signaling in regulating osteoclast function and bone resorption, and provide new therapeutic strategies for treatment and prevention of bone diseases such as osteoporosis.